An experimental study to assess the role of the goat in the decrease of virulence and transmission of Theileria lestoquardi

An experimental study to assess the role of the goat in the decrease of virulence and transmission of Theileria lestoquardi | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article An experimental study to assess the role of the goat in the decrease of virulence and transmission of Theileria lestoquardi Ali Jafarian Jelodar, Saeed Yaghfoori, Gholamreza Razmi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7574390/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Malignant Ovine Theileriosis (MOT) is a haemoprotozoan disease affecting sheep and goats, transmitted by the tick Hyalomma anatolicum anatolicum . The acute form is more commonly observed in sheep than in goats. This experimental study aimed to evaluate the effect of the goat on the virulence and transmission of T.lestoquardi . The experimental work was conducted between April 2020 and September 2021. In the first phase, sheep and goats were infected with T. lestoquardi via adult H. a. anatolicum ticks. Daily clinical and paraclinical examinations were performed, and infected animals were treated with buparvaquone (2.5 mg/kg) to prevent mortality. In the second phase, Theileria -free H.a. anatolicum nymphs were allowed to feed on the infected sheep and goats. The salivary glands of the emerged adult ticks were examined for T. lestoquardi using PCR. Four healthy, six-month-old sheep were divided into two groups. Each group was infected with 30 adult ticks (10 male, 20 female) originating from either ovine or caprine T. lestoquardi infections. Clinical signs were monitored daily, and blood and lymph node biopsy smears, as well as whole blood samples (in EDTA), were collected on days 0, 4, 8, 12, 14, 18, 22, 26, and 30 post-infection (PI) for paraclinical and molecular analysis. Initial clinical signs, such as fever and enlargement of the prescapular lymph nodes, appeared in both groups within 4–5 days PI. However, the clinical signs in Group 2 (infected with the ovine isolate) were significantly more severe. These sheep exhibited higher body temperatures, greater lymph node enlargement, and higher parasitemia levels compared to Group 1(infected with the caprine isolate) (P < 0.05). Piroplasms and schizonts of Theileria spp. were typically observed from days 8 and 13 PI, respectively. Transmission of T. lestoquardi to sheep was confirmed via PCR. Significant differences were observed between the two groups in packed cell volume (PCV), red blood cell (RBC) count, and white blood cell (WBC) count (P < 0.05). These findings indicate that the T. lestoquardi derived from a goat exhibited lower virulence than that derived from a sheep. The results suggest that goats may serve as potential carriers of T. lestoquardi . Experimental infection Hylomma anatolicm anatolicum Theileria lestoquardi Sheep Goat Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Malignant Ovine Theileriosis (MOT) is a tick-borne disease associated with high morbidity and mortality in sheep and goats across tropical and subtropical regions. (Uilenberg, 1995 ; Ahmed et al., 2011 ;Alessandra and Santo, 2012 ). Among Theileria species infecting small ruminants, T. lestoquardi , T. uilenbergi , and T. luwenshuni are considered highly pathogenic. In contrast, other species, T. Ovis , T. recondita , and T. separata are either weakly pathogenic or non-pathogenic(Uilenberg, 1997 ; Ahmed et al., 2011 ; Li YouQuan et al., 2012 ). T. lestoquardi , transmitted by Hyalomma anatolicum anatolicum , causes MOT(Hooshmand-Rad and Hawa, 1973 ;Taha and ElHussein, 2010 ). Sheep are highly susceptible, often developing subacute to acute infections even among indigenous breeds, whereas goats tend to show more resistance in endemic regions.(Brown et al., 1998 ; Kirvar et al., 1998 ). In Iran, sheep and goats are often raised together, and T. lestoquardi and T. ovis are the most frequently reported species (Hashemi-Fesharki, 1997 ;Razmi et al., 2003 ). One study showed that clinical signs of MOT were more severe in sheep than in goats in Iran (Hassan et al. 2015 ). However, the role of goats as carriers of MOT within sheep herds remains poorly understood. This study aimed to compare the clinical and paraclinical manifestations in sheep experimentally infected with T. lestoquardi derived from goats versus sheep. 2. Materials and Methods 2.1. Experimental Design Two experimental phases were carried out as detailed below: Phase 1-A involved intentionally infecting a sheep and a goat with T. lestoquardi and subsequently treating them with buparvaquone seven days post-infection to prevent mortality. Phase 2-Two months later, four sheep were separated into two groups and infected with T. lestoquardi obtained from the sheep and goat in Phase 1. Clinical and paraclinical parameters were analyzed and compared between the groups. 2.1.1. Phase 1 2.1.1.1. Animals and Pre-Infection Screening Six-month-old sheep and goats were purchased from a Theileria -free farm in Mashhad, Razavi Khorasan Province. Clinical examinations and semi-nested PCR confirmed they were free of piroplasms (Shayan and Rahbari 2005 ). Fecal samples were examined using parasitology methods(E. J. L. Soulsby 1982 ), and infected animals received anthelmintic treatment. Both animals and the experimental site were treated with acaricide. 2.1.1.2. Tick-mediated Infection Adult unfed H. a. anatolicum ticks infected with T. lestoquardi from the parasitology lab at Ferdowsi University of Mashhad were used for transmission (Yaghfoori et al., 2016 ; Yaghfoori et al., 2017 ). Thirty infected ticks (10 males, 20 females) were placed in ear bags. All ticks attached successfully and were fed completely. Animals were clinically examined daily, and lymph node aspirates and blood smears were collected to identify schizonts and assess parasitaemia. A body temperature of 41°C sustained for over three days was classified as lethal, prompting treatment with buparvaquone (2.5 mg/kg). 2.1.1.3. Production of H. a. anatolicum Nymphs New Zealand white rabbits were used to propagate H. a. anatolicum colonies(Tajeri and Razmi 2011 ). Approximately 1,500 larvae were reared inside ear bags affixed with zinc oxide paste. Larvae molted on the host, and newly emerged nymphs were removed 7–8 days post-infestation. Batches of 300 nymphs were stored in glass tubes in an incubator (28°C, 85% humidity, complete darkness). 2.1.1.4. Production of Infected Adult Ticks Two days later, 300 semi-engorged, uninfected nymphs were placed in ear bags on infected animals from Phase 1. After two weeks, engorged nymphs were collected and incubated under the same conditions. After 28 days, nymphs molted into adults. PCR analysis of their salivary glands confirmed infection with T. lestoquardi . 2.1.2. Phase 2 2.1.2.1 Adult Tick Transmission of T. lestoquardi Four six-month-old Baluchi sheep confirmed Theileria -free via clinical and PCR testing were purchased from the same farm. They were divided into two groups and infected with 30 adult ticks (10 males and 20 females) that had previously fed on Phase 1 sheep and goat, respectively.All ticks successfully fed on the sheep. Daily clinical monitoring included lymph node palpation and blood smear analysis for schizonts and parasitaemia. Blood samples were collected from the jugular vein into EDTA vacutainers on days 4, 8, 11, 13, 18, 22, 26, and 30 post-infection. 2.1.2.2. Hematological and parasitological Assessment Hematologic parameters—including RBC count, Hb concentration, PCV, and WBC count—were measured using an automatic veterinary cell counter (Celltac α, NEK–6450 K, Nihon Kohden, Tokyo, Japan). Parasitaemia was estimated by examining 100 microscopic fields of Giemsa-stained blood smears at a magnification of ×1000 (Razmi et al. 2003 ). 2.1.3. Statistical Analysis Data were analyzed using SPSS v22 (Chicago, USA) with the Friedman test. A p-value < 0.05 was considered statistically significant. 3. Results By day 12 post-infection (PI), the goat’s body temperature had risen to 39.5°C, whereas the sheep exhibited a higher temperature of 41.5°C. Enlargement of the pre-scapular lymph nodes was also observed in the infected goat (Fig. 1). Both piroplasm and schizont stages of Theileria spp. were detected in blood and lymph node smears from the infected sheep and goat (Figs. 2–3). On day 12 PI, parasitemia levels reached 2.0% in the goat and 2.8% in the sheep (Table 1). PCR analysis confirmed T. lestoquardi infection in the blood samples of both animals. To avoid mortality, animals showing signs of clinical distress were treated with Buparvaquone. The infected goat showed a better response to Buparvaquone treatment compared to the sheep. At three months PI, piroplasms were rarely detected in the goat’s blood smears, while they remained present in those of the sheep. Table 2 summarizes the main clinical, parasitological, and hematological findings in the two groups of sheep infected with T. lestoquardi . Both groups exhibited similar clinical signs, including fever, emaciation, enlargement of superficial lymph nodes, anemia, and icterus. However, the severity and duration of clinical symptoms were considerably greater in Group 1 than in Group 2. Piroplasms were first detected in blood smears on day 8 PI in both groups. Although the mean parasitemia percentages did not differ significantly between the two groups, the peak parasitemia was higher in Group 1(Fig. 4). Schizonts were identified in the superficial lymph nodes on day 8 PI in Group 1 and on day 13 PI in Group 2. Significant differences were observed between the two groups in hematological parameters, including hemoglobin (Hb) levels, packed cell volume (PCV), red blood cell (RBC) count (Table 2, Fig. 5) (p < 0.05), white blood cell (WBC) count, and differential counts of neutrophils and lymphocytes (Table 2, Fig. 6). After 3 months post-infection, it was difficult to find piroplasms in the goat blood smear, while they were still detectable in the blood smear of the infected sheep. Table 1 Comparison of the clinical and parasitological parameters in infected sheep and goat Clinical and parasitological parameters Sheep Goat First day of fever > 39.7 (˚C) 3 3 Maximum fever (˚C) 41.5 39.9 First day of piroplasm detection 6 8 First day lymph node enlargement 3 3 Table 2 clinical and paraclinical parameters in ovine and caprine theileriosis (Mean ± SD) Parameters Group 1 Group2 Reference range P-value Clinical signs First day of fever > 39.8C 8 8 - Total number of days with fever 5 3 - Max. fever (˚C) 41.5 40 - The range of fever (Mean ± SD) 40.16 ± 0.89 39.26 ± o.52 - P < 0.008 Parasitology First day of schizont detection in the lymph node 8 13 - First day of piroplasm detection 8 8 - Peak piroplasm parasitaemia (%) 3.5 0.25 - The day of peak piroplasm parasitaemia 18 18 - The range of parasitaemia (Mean ± SD) 0.54 ± 1.11 0.15 ± 011 - Haematology Parameters (Mean ± SD) Packed cell volume (%) 17.83 ± 3.82 23.25 ± 1.98 27–45 P < 0.001 Red blood cells (×10 6 /dL) 6.82 ± 1.68 9.08 ± 0.68 9–15 P < 0.07 Haemoglobin (g/dL) 7.51 ± 1.5 9.5 ± 0.71 9–15 P < 0.002 White blood cells (cells/mm 3 ) 11677 ± 3003 7994 ± 1623 4000–12000 P < 0.003 Neutrophil 6890 ± 1992 4385 ± 1286 700–6000 P < 0.02 Lymphocyte 4033 ± 1554 3274 ± 649 2000–9000 P < 0.04 Monocyte 302 ± 304 133 ± 116 0-750 P < 0.2 Eosinophil 134 ± 318 253 ± 220 0-1000 P < 0.04 4. Discussion An experimental study was conducted to assess the role of goats in the transmission of T.lestoquardi infection among herd sheep. In this study, T. lestoquardi was experimentally transmitted to sheep and goat using H. a. anatolicum ticks. Infected sheep developed higher fever and parasitemia than infected goats. A similar study demonstrated that T. lestoquardi infected both hosts, causing moderate to severe clinical reactions in sheep and only mild to moderate signs in goats (Brown et al., 1998 ). Another experiment showed that all sheep were readily infected, while goats appeared less susceptible to piroplasms, and schizonts were detected in only one out of four goats (Kirvar et al., 1998 ). Goats generally appear more resistant to T. lestoquardi than sheep, typically exhibiting only mild to moderate clinical signs (Brown et al., 1998 ; Hassan et al., 2015 ; Shruthi et al., 2017; Stuen, 2016; Rathod et al., 2021). However, an outbreak of malignant ovine theileriosis resulting in significant losses among imported goats in Sudan has been documented(Taha et al. 2011 ). The successful acquisition of T. lestoquardi from infected sheep and goats by nymphal H. a. anatolicum ticks, and its subsequent transmission to sheep by adult ticks, confirmed that goats, like sheep, can act as a carrier of disease (Ahmed et al., 2011 ). In Phase 2 of the current study, both sheep groups exhibited clinical signs of malignant ovine theileriosis (MOT). However, symptoms were more severe in Group 1, which was infected with ovine-derived T. lestoquardi , than in Group 2, infected with caprine-derived parasites. Group 1 developed severe anemia, whereas Group 2 showed no signs of anemia. Hematological parameters, including RBC count, hemoglobin (Hb), and packed cell volume (PCV), were significantly lower in Group 1 compared to Group 2. Both groups initially developed leukocytosis, followed by a decline in white blood cell (WBC) counts toward the end of the study, with Group 1 exhibiting a more pronounced and gradual leukopenia. Previous studies have reported declines in hematological values in T. lestoquardi -infected sheep under experimental conditions (Elsadig et al., 2013; Yaghfoori et al., 2017 ; Razmi et al., 2019). Epidemiological studies also revealed reduced RBCs, Hb, and PCV in infected herd sheep (Hassan et al., 2015 ; Riaz and Tasawar, 2017). Based on current findings, T. lestoquardi was successfully transmitted from goats to sheep, resulting in mild MOT. It remains uncertain whether the reduced virulence of goat-derived T. lestoquardi is due to genetic mutations or a lower dose of sporozoites during inoculation. Further comparative studies using quantified doses of fresh isolates or cloned parasites are necessary to test this hypothesis. A comprehensive genetic diversity assessment of T. lestoquardi in Sudanese sheep showed high variability, with most isolates from both sheep and goats clustering together (Ali et al., 2017). In conclusion, while goats can develop mild clinical theileriosis, they may be nearly as susceptible as sheep. Importantly, they can serve as reservoir hosts for T. lestoquardi , facilitating transmission to sheep and contributing to subacute or covert disease manifestations. Declarations Ethics approval and consent to participate: All experiments involving animals were conducted in full compliance with the guidelines sanctioned by the Animal Ethics Committee of our faculty, reference number IR.UM.REC.1398.070 Consent for publication: Not applicable Availability of data and materials: Not applicable Competing interests: The authors declare that they have no competing interests in this section. Funding: This study was supported by grant 3/56889 from the Vice President of Research and Technology of Veterinary Medicine, Ferdowsi University of Mashhad, Iran. Author Contributions: G.R.R : design of the work; A.J, S.Y, G.R.R: the acquisition, analysis, A.J, S.Y: Interpretation of data; A.J :The creation of new software used in the work; A.J, G.R.R : Writing - original draft preparation ; G.R.R,S.Y: Review and editing; G.R.R: Funding acquisition and Supervision Acknowledgments: We want to express our gratitude to Mr. Hamid Eshrati and Mr. Mohammad Nejad for their assistance with the laboratory examination. References Ahmed J, Yin H, Bakheit M, et al (2011) Small Ruminant Theileriosis. In: Mehlhorn H (ed) Progress in Parasitology. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 135–153 Alessandra T, Santo C (2012) Tick-borne diseases in sheep and goats: Clinical and diagnostic aspects. Small Rumin Res 106:S6–S11. https://doi.org/https://doi.org/10.1016/j.smallrumres.2012.04.026 Brown CGD, Ilhan T, Kirvar E, et al (1998) Theileria lestoquardi and T. annulata in Cattle, Sheep, and Goats: In Vitro and in Vivo Studies. Ann N Y Acad Sci 849:44–51. https://doi.org/https://doi.org/10.1111/j.1749-6632.1998.tb11032.x E. J. L. Soulsby (1982) Helminths, arthropods and protozoa of domesticated animals No Title. Baillière Tindall, London Hashemi-Fesharki R (1997) Tick-borne diseases of sheep and goats and their related vectors in Iran. Parassitologia 39:115–117 Hassan MA, Raoofi A, Lotfollahzadeh S, Javanbakht J (2015) Clinical and cytological characteristics and prognostic implications on sheep and goat Theileria infection in north of Iran. J Parasit Dis 39:190–193. https://doi.org/10.1007/s12639-013-0318-1 Hooshmand-Rad P, Hawa NJ (1973) Malignant theileriosis of sheep and goats. 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Elsevier, pp 55–61 Yaghfoori S, Razmi GR, Mohri M, et al (2016) An experimental ovine Theileriosis: The effect of Theileria lestoquardi infection on cardiovascular system in sheep. Acta Trop 161:. https://doi.org/10.1016/j.actatropica.2016.05.014 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7574390","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516883274,"identity":"18f9f809-a0d4-4fc2-84aa-caa1a0ac5dbb","order_by":0,"name":"Ali Jafarian Jelodar","email":"","orcid":"","institution":"Ferdowsi University of Mashhad","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Jafarian","lastName":"Jelodar","suffix":""},{"id":516883275,"identity":"474e9073-f1d9-426a-830b-0012ca38969a","order_by":1,"name":"Saeed Yaghfoori","email":"","orcid":"","institution":"Ferdowsi University of 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06:12:36","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74816,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7574390/v1/88f854288856b7dd9f61ca16.html"},{"id":92052322,"identity":"c9e1afad-b4a1-4d8f-ad76-620df701616d","added_by":"auto","created_at":"2025-09-24 06:12:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":214785,"visible":true,"origin":"","legend":"\u003cp\u003eLymph node enlargement in an infected goat with \u003cem\u003eT.lestoquardi\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.1infectedgoat.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7574390/v1/70a717e9ab0d7f2a0a526c4c.jpg"},{"id":92052312,"identity":"c48fd7ee-d2e7-4145-a496-b22c3ba9e72c","added_by":"auto","created_at":"2025-09-24 06:12:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79421,"visible":true,"origin":"","legend":"\u003cp\u003eA-Blood smears showing piroplasm forms (arrows) of \u003cem\u003eT. lestoquardi\u003c/em\u003e in the red blood cells、Giemsa stain, ×1000. B- Lymph node smears displaying a schizont (arrow) in a mononuclear cell. Giemsa stain, ×1000\u003c/p\u003e","description":"","filename":"Fig.2PiroplasmSchizont.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7574390/v1/f868136749193813d611e6b3.jpg"},{"id":92052909,"identity":"7fcbaa3b-9e89-4f23-ae50-e152955573fc","added_by":"auto","created_at":"2025-09-24 06:20:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43726,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of fever and parasitemia rate between infected sheep and goat\u003c/p\u003e","description":"","filename":"Fig.3Phase2Parasitemia.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7574390/v1/edafe8db7cd9a663bfa1251d.jpg"},{"id":92052315,"identity":"939286de-35b1-4fc7-be72-3f033a825930","added_by":"auto","created_at":"2025-09-24 06:12:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":110868,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of parasitemia rate between infected sheep in two groups\u003c/p\u003e","description":"","filename":"Fig.4RBCHbPCV.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7574390/v1/762e985516ca6a90bfdd88ef.jpg"},{"id":92052318,"identity":"e8dc6283-365e-44da-b20b-d7c9fe697c6a","added_by":"auto","created_at":"2025-09-24 06:12:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":117402,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of RBC, Hb, and PCV count between infected sheep in two groups\u003c/p\u003e","description":"","filename":"Fig.5WBClymphocyteneutrophil.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7574390/v1/f88ccd870cb2d16711d2db86.jpg"},{"id":92052910,"identity":"3aa41add-2caa-4f2d-8b91-e9e1ee42c47a","added_by":"auto","created_at":"2025-09-24 06:20:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11975,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of WBC, Lymphocytes, and neutrophil counts between infected sheep in two groups\u003c/p\u003e","description":"","filename":"fig.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7574390/v1/2d4dd744c3abdcc3f603c5d7.jpg"},{"id":93465399,"identity":"22398574-07bc-4b5c-b651-3311cdc10c38","added_by":"auto","created_at":"2025-10-14 07:17:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1308520,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7574390/v1/c5fe48b3-0ba3-4525-9af5-38042b6f6c6e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"An experimental study to assess the role of the goat in the decrease of virulence and transmission of Theileria lestoquardi","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMalignant Ovine Theileriosis (MOT) is a tick-borne disease associated with high morbidity and mortality in sheep and goats across tropical and subtropical regions. (Uilenberg, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Ahmed et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e;Alessandra and Santo, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Among Theileria species infecting small ruminants, \u003cem\u003eT. lestoquardi\u003c/em\u003e, \u003cem\u003eT. uilenbergi\u003c/em\u003e, and \u003cem\u003eT. luwenshuni\u003c/em\u003e are considered highly pathogenic. In contrast, other species, \u003cem\u003eT. Ovis\u003c/em\u003e, \u003cem\u003eT. recondita\u003c/em\u003e, and \u003cem\u003eT. separata\u003c/em\u003e are either weakly pathogenic or non-pathogenic(Uilenberg, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Ahmed et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Li YouQuan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003eT. lestoquardi\u003c/em\u003e, transmitted by \u003cem\u003eHyalomma anatolicum anatolicum\u003c/em\u003e, causes MOT(Hooshmand-Rad and Hawa, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1973\u003c/span\u003e;Taha and ElHussein, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Sheep are highly susceptible, often developing subacute to acute infections even among indigenous breeds, whereas goats tend to show more resistance in endemic regions.(Brown et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Kirvar et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In Iran, sheep and goats are often raised together, and \u003cem\u003eT. lestoquardi\u003c/em\u003e and \u003cem\u003eT. ovis\u003c/em\u003e are the most frequently reported species (Hashemi-Fesharki, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1997\u003c/span\u003e;Razmi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). One study showed that clinical signs of MOT were more severe in sheep than in goats in Iran (Hassan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, the role of goats as carriers of MOT within sheep herds remains poorly understood. This study aimed to compare the clinical and paraclinical manifestations in sheep experimentally infected with \u003cem\u003eT. lestoquardi\u003c/em\u003e derived from goats versus sheep.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Experimental Design\u003c/h2\u003e\u003cp\u003eTwo experimental phases were carried out as detailed below: Phase 1-A involved intentionally infecting a sheep and a goat with T. lestoquardi and subsequently treating them with buparvaquone seven days post-infection to prevent mortality. Phase 2-Two months later, four sheep were separated into two groups and infected with \u003cem\u003eT. lestoquardi\u003c/em\u003e obtained from the sheep and goat in Phase 1. Clinical and paraclinical parameters were analyzed and compared between the groups.\u003c/p\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1. Phase 1\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section4\"\u003e\u003ch2\u003e2.1.1.1. Animals and Pre-Infection Screening\u003c/h2\u003e\u003cp\u003eSix-month-old sheep and goats were purchased from a \u003cem\u003eTheileria\u003c/em\u003e-free farm in Mashhad, Razavi Khorasan Province. Clinical examinations and semi-nested PCR confirmed they were free of piroplasms (Shayan and Rahbari \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Fecal samples were examined using parasitology methods(E. J. L. Soulsby \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1982\u003c/span\u003e), and infected animals received anthelmintic treatment. Both animals and the experimental site were treated with acaricide.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section4\"\u003e\u003ch2\u003e2.1.1.2. Tick-mediated Infection\u003c/h2\u003e\u003cp\u003eAdult unfed \u003cem\u003eH. a. anatolicum\u003c/em\u003e ticks infected with \u003cem\u003eT. lestoquardi\u003c/em\u003e from the parasitology lab at Ferdowsi University of Mashhad were used for transmission (Yaghfoori et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yaghfoori et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thirty infected ticks (10 males, 20 females) were placed in ear bags. All ticks attached successfully and were fed completely. Animals were clinically examined daily, and lymph node aspirates and blood smears were collected to identify schizonts and assess parasitaemia. A body temperature of 41\u0026deg;C sustained for over three days was classified as lethal, prompting treatment with buparvaquone (2.5 mg/kg).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section4\"\u003e\u003ch2\u003e2.1.1.3. Production of \u003cem\u003eH. a. anatolicum\u003c/em\u003e Nymphs\u003c/h2\u003e\u003cp\u003eNew Zealand white rabbits were used to propagate \u003cem\u003eH. a. anatolicum\u003c/em\u003e colonies(Tajeri and Razmi \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Approximately 1,500 larvae were reared inside ear bags affixed with zinc oxide paste. Larvae molted on the host, and newly emerged nymphs were removed 7\u0026ndash;8 days post-infestation. Batches of 300 nymphs were stored in glass tubes in an incubator (28\u0026deg;C, 85% humidity, complete darkness).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section4\"\u003e\u003ch2\u003e2.1.1.4. Production of Infected Adult Ticks\u003c/h2\u003e\u003cp\u003eTwo days later, 300 semi-engorged, uninfected nymphs were placed in ear bags on infected animals from Phase 1. After two weeks, engorged nymphs were collected and incubated under the same conditions. After 28 days, nymphs molted into adults. PCR analysis of their salivary glands confirmed infection with \u003cem\u003eT. lestoquardi\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.1.2. Phase 2\u003c/h2\u003e\u003cdiv id=\"Sec10\" class=\"Section4\"\u003e\u003ch2\u003e2.1.2.1 Adult Tick Transmission of \u003cem\u003eT. lestoquardi\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eFour six-month-old Baluchi sheep confirmed \u003cem\u003eTheileria\u003c/em\u003e-free via clinical and PCR testing were purchased from the same farm. They were divided into two groups and infected with 30 adult ticks (10 males and 20 females) that had previously fed on Phase 1 sheep and goat, respectively.All ticks successfully fed on the sheep. Daily clinical monitoring included lymph node palpation and blood smear analysis for schizonts and parasitaemia. Blood samples were collected from the jugular vein into EDTA vacutainers on days 4, 8, 11, 13, 18, 22, 26, and 30 post-infection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section4\"\u003e\u003ch2\u003e2.1.2.2. Hematological and parasitological Assessment\u003c/h2\u003e\u003cp\u003eHematologic parameters\u0026mdash;including RBC count, Hb concentration, PCV, and WBC count\u0026mdash;were measured using an automatic veterinary cell counter (Celltac α, NEK\u0026ndash;6450 K, Nihon Kohden, Tokyo, Japan). Parasitaemia was estimated by examining 100 microscopic fields of Giemsa-stained blood smears at a magnification of \u0026times;1000 (Razmi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.1.3. Statistical Analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using SPSS v22 (Chicago, USA) with the Friedman test. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eBy day 12 post-infection (PI), the goat\u0026rsquo;s body temperature had risen to 39.5\u0026deg;C, whereas the sheep exhibited a higher temperature of 41.5\u0026deg;C. Enlargement of the pre-scapular lymph nodes was also observed in the infected goat (Fig. 1). Both piroplasm and schizont stages of \u003cem\u003eTheileria\u003c/em\u003e spp. were detected in blood and lymph node smears from the infected sheep and goat (Figs. 2\u0026ndash;3). On day 12 PI, parasitemia levels reached 2.0% in the goat and 2.8% in the sheep (Table 1). PCR analysis confirmed \u003cem\u003eT. lestoquardi\u003c/em\u003e infection in the blood samples of both animals. To avoid mortality, animals showing signs of clinical distress were treated with Buparvaquone. The infected goat showed a better response to Buparvaquone treatment compared to the sheep. At three months PI, piroplasms were rarely detected in the goat\u0026rsquo;s blood smears, while they remained present in those of the sheep. Table 2 summarizes the main clinical, parasitological, and hematological findings in the two groups of sheep infected with \u003cem\u003eT. lestoquardi\u003c/em\u003e. Both groups exhibited similar clinical signs, including fever, emaciation, enlargement of superficial lymph nodes, anemia, and icterus. However, the severity and duration of clinical symptoms were considerably greater in Group 1 than in Group 2. Piroplasms were first detected in blood smears on day 8 PI in both groups. Although the mean parasitemia percentages did not differ significantly between the two groups, the peak parasitemia was higher in Group 1(Fig. 4). Schizonts were identified in the superficial lymph nodes on day 8 PI in Group 1 and on day 13 PI in Group 2. Significant differences were observed between the two groups in hematological parameters, including hemoglobin (Hb) levels, packed cell volume (PCV), red blood cell (RBC) count (Table 2, Fig. 5) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), white blood cell (WBC) count, and differential counts of neutrophils and lymphocytes (Table 2, Fig. 6). After 3 months post-infection, it was difficult to find piroplasms in the goat blood smear, while they were still detectable in the blood smear of the infected sheep.\u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eComparison of the clinical and parasitological parameters in infected sheep and goat\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClinical and parasitological parameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSheep\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFirst day of fever\u0026thinsp;\u0026gt;\u0026thinsp;39.7 (˚C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMaximum fever (˚C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFirst day of piroplasm detection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFirst day lymph node enlargement\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eclinical and paraclinical parameters in ovine and caprine theileriosis (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReference range\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical signs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFirst day of fever\u0026thinsp;\u0026gt;\u0026thinsp;39.8C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal number of days with fever\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMax. fever (˚C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe range of fever\u003c/p\u003e\n \u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.26\u0026thinsp;\u0026plusmn;\u0026thinsp;o.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003eParasitology\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFirst day of schizont detection in the lymph node\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFirst day of piroplasm detection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePeak piroplasm parasitaemia (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe day of peak piroplasm parasitaemia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe range of parasitaemia\u003c/p\u003e\n \u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"8\"\u003e\n \u003cp\u003e\u003cstrong\u003eHaematology\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePacked cell volume (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u0026ndash;45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRed blood cells (\u0026times;10\u003csup\u003e6\u003c/sup\u003e/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u0026ndash;15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHaemoglobin (g/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u0026ndash;15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWhite blood cells (cells/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11677\u0026thinsp;\u0026plusmn;\u0026thinsp;3003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7994\u0026thinsp;\u0026plusmn;\u0026thinsp;1623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4000\u0026ndash;12000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeutrophil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6890\u0026thinsp;\u0026plusmn;\u0026thinsp;1992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4385\u0026thinsp;\u0026plusmn;\u0026thinsp;1286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e700\u0026ndash;6000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLymphocyte\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4033\u0026thinsp;\u0026plusmn;\u0026thinsp;1554\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3274\u0026thinsp;\u0026plusmn;\u0026thinsp;649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2000\u0026ndash;9000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMonocyte\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e302\u0026thinsp;\u0026plusmn;\u0026thinsp;304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e133\u0026thinsp;\u0026plusmn;\u0026thinsp;116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0-750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEosinophil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134\u0026thinsp;\u0026plusmn;\u0026thinsp;318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e253\u0026thinsp;\u0026plusmn;\u0026thinsp;220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0-1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"},{"header":"4. Discussion","content":"\u003cp\u003eAn experimental study was conducted to assess the role of goats in the transmission of \u003cem\u003eT.lestoquardi\u003c/em\u003e infection among herd sheep. In this study, \u003cem\u003eT. lestoquardi\u003c/em\u003e was experimentally transmitted to sheep and goat using \u003cem\u003eH. a. anatolicum\u003c/em\u003e ticks. Infected sheep developed higher fever and parasitemia than infected goats. A similar study demonstrated that \u003cem\u003eT. lestoquardi\u003c/em\u003e infected both hosts, causing moderate to severe clinical reactions in sheep and only mild to moderate signs in goats (Brown et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Another experiment showed that all sheep were readily infected, while goats appeared less susceptible to piroplasms, and schizonts were detected in only one out of four goats (Kirvar et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Goats generally appear more resistant to \u003cem\u003eT. lestoquardi\u003c/em\u003e than sheep, typically exhibiting only mild to moderate clinical signs (Brown et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Hassan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shruthi et al., 2017; Stuen, 2016; Rathod et al., 2021). However, an outbreak of malignant ovine theileriosis resulting in significant losses among imported goats in Sudan has been documented(Taha et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The successful acquisition of \u003cem\u003eT. lestoquardi\u003c/em\u003e from infected sheep and goats by nymphal \u003cem\u003eH. a. anatolicum\u003c/em\u003e ticks, and its subsequent transmission to sheep by adult ticks, confirmed that goats, like sheep, can act as a carrier of disease (Ahmed et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In Phase 2 of the current study, both sheep groups exhibited clinical signs of malignant ovine theileriosis (MOT). However, symptoms were more severe in Group 1, which was infected with ovine-derived \u003cem\u003eT. lestoquardi\u003c/em\u003e, than in Group 2, infected with caprine-derived parasites. Group 1 developed severe anemia, whereas Group 2 showed no signs of anemia. Hematological parameters, including RBC count, hemoglobin (Hb), and packed cell volume (PCV), were significantly lower in Group 1 compared to Group 2. Both groups initially developed leukocytosis, followed by a decline in white blood cell (WBC) counts toward the end of the study, with Group 1 exhibiting a more pronounced and gradual leukopenia. Previous studies have reported declines in hematological values in \u003cem\u003eT. lestoquardi\u003c/em\u003e-infected sheep under experimental conditions (Elsadig et al., 2013; Yaghfoori et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Razmi et al., 2019). Epidemiological studies also revealed reduced RBCs, Hb, and PCV in infected herd sheep (Hassan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Riaz and Tasawar, 2017). Based on current findings, \u003cem\u003eT. lestoquardi\u003c/em\u003e was successfully transmitted from goats to sheep, resulting in mild MOT. It remains uncertain whether the reduced virulence of goat-derived \u003cem\u003eT. lestoquardi\u003c/em\u003e is due to genetic mutations or a lower dose of sporozoites during inoculation. Further comparative studies using quantified doses of fresh isolates or cloned parasites are necessary to test this hypothesis. A comprehensive genetic diversity assessment of \u003cem\u003eT. lestoquardi\u003c/em\u003e in Sudanese sheep showed high variability, with most isolates from both sheep and goats clustering together (Ali et al., 2017). In conclusion, while goats can develop mild clinical theileriosis, they may be nearly as susceptible as sheep. Importantly, they can serve as reservoir hosts for \u003cem\u003eT. lestoquardi\u003c/em\u003e, facilitating transmission to sheep and contributing to subacute or covert disease manifestations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eAll experiments involving animals were conducted in full compliance with the guidelines sanctioned by the Animal Ethics Committee of our faculty, reference number IR.UM.REC.1398.070\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests in this section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by grant 3/56889 from the Vice President of Research and Technology of Veterinary Medicine, Ferdowsi University of Mashhad, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eG.R.R :\u003c/strong\u003e design of the work; \u003cstrong\u003eA.J, S.Y, G.R.R:\u003c/strong\u003e the acquisition, analysis, \u003cstrong\u003eA.J, S.Y:\u003c/strong\u003e Interpretation of data; \u003cstrong\u003eA.J\u003c/strong\u003e :The creation of new software used in the work; \u003cstrong\u003eA.J, G.R.R\u003c/strong\u003e : Writing - original draft preparation\u003cstrong\u003e; G.R.R,S.Y:\u0026nbsp;\u003c/strong\u003eReview and editing;\u003cstrong\u003e\u0026nbsp;G.R.R:\u0026nbsp;\u003c/strong\u003eFunding acquisition\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand Supervision\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to express our gratitude to Mr. Hamid Eshrati and Mr. Mohammad Nejad for their assistance with the laboratory examination. \u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmed J, Yin H, Bakheit M, et al (2011) Small Ruminant Theileriosis. In: Mehlhorn H (ed) Progress in Parasitology. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 135\u0026ndash;153\u003c/li\u003e\n\u003cli\u003eAlessandra T, Santo C (2012) Tick-borne diseases in sheep and goats: Clinical and diagnostic aspects. Small Rumin Res 106:S6\u0026ndash;S11. https://doi.org/https://doi.org/10.1016/j.smallrumres.2012.04.026\u003c/li\u003e\n\u003cli\u003eBrown CGD, Ilhan T, Kirvar E, et al (1998) Theileria lestoquardi and T. annulata in Cattle, Sheep, and Goats: In Vitro and in Vivo Studies. Ann N Y Acad Sci 849:44\u0026ndash;51. https://doi.org/https://doi.org/10.1111/j.1749-6632.1998.tb11032.x\u003c/li\u003e\n\u003cli\u003eE. J. L. Soulsby (1982) Helminths, arthropods and protozoa of domesticated animals No Title. Bailli\u0026egrave;re Tindall, London\u003c/li\u003e\n\u003cli\u003eHashemi-Fesharki R (1997) Tick-borne diseases of sheep and goats and their related vectors in Iran. Parassitologia 39:115\u0026ndash;117\u003c/li\u003e\n\u003cli\u003eHassan MA, Raoofi A, Lotfollahzadeh S, Javanbakht J (2015) Clinical and cytological characteristics and prognostic implications on sheep and goat Theileria infection in north of Iran. J Parasit Dis 39:190\u0026ndash;193. https://doi.org/10.1007/s12639-013-0318-1\u003c/li\u003e\n\u003cli\u003eHooshmand-Rad P, Hawa NJ (1973) Malignant theileriosis of sheep and goats. Trop Anim Health Prod 5:97\u0026ndash;102\u003c/li\u003e\n\u003cli\u003eKirvar E, Ilhan T, Katzer F, et al (1998) Detection of Theileria lestoquardi (hirci) in Ticks, Sheep, and Goats Using the Polymerase Chain Reaction. Ann N Y Acad Sci 849:52\u0026ndash;62. https://doi.org/https://doi.org/10.1111/j.1749-6632.1998.tb11033.x\u003c/li\u003e\n\u003cli\u003eLi YouQuan LY, Peng YuLu PY, Liu ZhiJie LZ, et al (2012) Epidemiological survey and identification of Theileria parasite infection for small ruminants in some parts of China. Sci Agric Sin 45:3422\u0026ndash;29\u003c/li\u003e\n\u003cli\u003eRazmi GR, Hosseini M, Aslani MR (2003) Identification of tick vectors of ovine theileriosis in an endemic region of Iran. Vet Parasitol 116:. https://doi.org/10.1016/S0304-4017(03)00254-1\u003c/li\u003e\n\u003cli\u003eShayan P, Rahbari S (2005) Simultaneous differentiation between Theileria spp. and Babesia spp. on stained blood smear using PCR. Parasitol Res 97:281\u0026ndash;286. https://doi.org/10.1007/s00436-005-1434-3\u003c/li\u003e\n\u003cli\u003eTaha KM, ElHussein AM (2010) Experimental transmission of Theileria lestoquardi by developmental stages of Hyalomma anatolicum ticks. Parasitol Res 107:1009\u0026ndash;1012. https://doi.org/10.1007/s00436-010-1968-x\u003c/li\u003e\n\u003cli\u003eTaha KM, Salih DA, Ahmed BM, et al (2011) First confirmed report of outbreak of malignant ovine theileriosis among goats in Sudan. Parasitol Res 109:1525\u0026ndash;1527. https://doi.org/10.1007/s00436-011-2428-y\u003c/li\u003e\n\u003cli\u003eTajeri S, Razmi GR (2011) Hyalomma anatolicum anatolicum and Hyalomma dromedarii (Acari: Ixodidae) imbibe bovine blood in vitro by utilizing an artificial feeding system. Vet Parasitol 180:. https://doi.org/10.1016/j.vetpar.2011.03.014\u003c/li\u003e\n\u003cli\u003eUilenberg G (1997) General review of tick-borne diseases of sheep and goats world-wide. Parassitologia 39:161\u0026ndash;165\u003c/li\u003e\n\u003cli\u003eUilenberg G (1995) International collaborative research: significance of tick-borne hemoparasitic diseases to world animal health. Vet Parasitol 57:19\u0026ndash;41. https://doi.org/10.1016/0304-4017(94)03107-8\u003c/li\u003e\n\u003cli\u003eYaghfoori S, Mohri M, Razmi G (2017) Experimental Theileria lestoquardi infection in sheep: Biochemical and hematological changes. In: Acta Tropica. Elsevier, pp 55\u0026ndash;61\u003c/li\u003e\n\u003cli\u003eYaghfoori S, Razmi GR, Mohri M, et al (2016) An experimental ovine Theileriosis: The effect of Theileria lestoquardi infection on cardiovascular system in sheep. Acta Trop 161:. https://doi.org/10.1016/j.actatropica.2016.05.014\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Experimental infection, Hylomma anatolicm anatolicum, Theileria lestoquardi, Sheep, Goat","lastPublishedDoi":"10.21203/rs.3.rs-7574390/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7574390/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMalignant Ovine Theileriosis (MOT) is a haemoprotozoan disease affecting sheep and goats, transmitted by the tick \u003cem\u003eHyalomma anatolicum anatolicum\u003c/em\u003e. The acute form is more commonly observed in sheep than in goats. This experimental study aimed to evaluate the effect of the goat on the virulence and transmission of \u003cem\u003eT.lestoquardi\u003c/em\u003e. The experimental work was conducted between April 2020 and September 2021. In the first phase, sheep and goats were infected with \u003cem\u003eT. lestoquardi\u003c/em\u003e via adult \u003cem\u003eH. a. anatolicum\u003c/em\u003e ticks. Daily clinical and paraclinical examinations were performed, and infected animals were treated with buparvaquone (2.5 mg/kg) to prevent mortality. In the second phase, \u003cem\u003eTheileria\u003c/em\u003e-free \u003cem\u003eH.a. anatolicum\u003c/em\u003e nymphs were allowed to feed on the infected sheep and goats. The salivary glands of the emerged adult ticks were examined for \u003cem\u003eT. lestoquardi\u003c/em\u003e using PCR. Four healthy, six-month-old sheep were divided into two groups. Each group was infected with 30 adult ticks (10 male, 20 female) originating from either ovine or caprine \u003cem\u003eT. lestoquardi\u003c/em\u003e infections. Clinical signs were monitored daily, and blood and lymph node biopsy smears, as well as whole blood samples (in EDTA), were collected on days 0, 4, 8, 12, 14, 18, 22, 26, and 30 post-infection (PI) for paraclinical and molecular analysis. Initial clinical signs, such as fever and enlargement of the prescapular lymph nodes, appeared in both groups within 4\u0026ndash;5 days PI. However, the clinical signs in Group 2 (infected with the ovine isolate) were significantly more severe. These sheep exhibited higher body temperatures, greater lymph node enlargement, and higher parasitemia levels compared to Group 1(infected with the caprine isolate) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Piroplasms and schizonts of \u003cem\u003eTheileria\u003c/em\u003e spp. were typically observed from days 8 and 13 PI, respectively. Transmission of \u003cem\u003eT. lestoquardi\u003c/em\u003e to sheep was confirmed via PCR. Significant differences were observed between the two groups in packed cell volume (PCV), red blood cell (RBC) count, and white blood cell (WBC) count (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicate that the \u003cem\u003eT. lestoquardi\u003c/em\u003e derived from a goat exhibited lower virulence than that derived from a sheep. The results suggest that goats may serve as potential carriers of \u003cem\u003eT. lestoquardi\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"An experimental study to assess the role of the goat in the decrease of virulence and transmission of Theileria lestoquardi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 06:12:31","doi":"10.21203/rs.3.rs-7574390/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3cdff92-d676-46ef-aaa8-74b06b892565","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-14T07:08:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 06:12:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7574390","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7574390","identity":"rs-7574390","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}